The pH of the intracellular compartment is tightly controlled in all eukaryotic cells. This control is crucial for various biological processes, including intracellular membrane trafficking, endocytosis, protein degradation, bone resorption, and small-molecule uptake. The vacuolar H+ ATPase (V-ATPase) is one of the central players in regulating acidity in eukaryotic cells and the loss of V-ATPase function is in general lethal at early stages of development.
V-ATPases are large multi-subunit protein complexes that function as a rotary molecular motor, and are organized into two domains, V0 and V1. The V1 domain is located on the cytoplasmic side of the membrane and carries out ATP hydrolysis, whereas the V0 domain is a membrane embedded complex that is responsible for proton translocation across the membrane.1-3 The V1 domain is composed of eight different subunits (A, B, C, D, E, F, G, H) and the V0 domain contains five different subunits (a, b, c, d, and e) in mammals, some of which are present in multiple copies. The core of the V1 domain contains a hexamer of A and B subunits, which participates in ATP binding and hydrolysis with the most of the residues responsible for ATP binding contributed by the catalytic subunit A.
Dysregulation of V-ATPase has been implicated in a number of diseases, including renal disease (renal tubular acidosis)4, bone disease (osteoporosis)5, and tumor metastasis.6 For example, the V-ATPase activity has been found to be significantly higher in the highly invasive MB231 breast cancer cells than the largely non-metastatic MCF7 cells.6,7 The treatment of MB231 cells with bafilomycin, a known V-ATPase inhibitor, significantly inhibited the invasiveness of cancer cells, suggesting that V-ATPase is a potential drug target for blocking cancer metastasis.2,8 In addition to cancer, V-ATPase is also implicated in renal and bone di seases.2,9 
A number of inhibitors of V-ATPase have been identified and tested for their therapeutic potentials. For example, macrolide antibiotics with 18-membered lactone rings, bafilomycin and concanamycin, were found to be selective inhibitors of V-ATPases soon after their isolation from Streptomyces in the 1980s.10 A series of studies revealed that these plecomacrolides primary bound to the V0 subunit c and perturbed rotation of the b/c-ring. In addition to inhibiting V-ATPase, bafilomycin impairs mitochondrial function by acting as a carrier type potassium ionophore.11 
Additional inhibitors of V-ATPase were subsequently discovered,10 such as archazolid, which is a natural product produced by the myxobacteria Archangium gephyra and Cystobacter violaceus. Archazolid, which also binds to c subunit, appears to be a highly potent V-ATPase inhibitor and blocks the growth of mammalian cells at subnanomolar concentrations. Another class of V-ATPase inhibitors, benzolactone enamides (e.g., salicylihamide, apicularens and cruentaren) that was isolated from various natural sources, demonstrated potent inhibition against mammalian V-ATPase, but surprisingly no effects on fungal V-ATPase. However, these natural products tend to be highly toxic to mammalian cells. Previous studies revealed that the binding sites of benzolactone enamides should still reside within V0 domain but differ from plecomacrolides. A number of novel indole derivatives were synthesized based on bafilomycin structure. Among these indole-containing bafilomycin analogs, INDOL0 interacts with the V0 subunit c and cause potent inhibition against V-ATPase. A number of V-ATPase inhibitors such as synthetic benzolactone enamide RTA203 (a derivative of salicylihalamide), indole derivatives NiK12192 and SB24278, tributyltin chloride (TBTCl), 3-bromopyruvate (3-Br-PA) have been reported recently. However, the binding site of these synthetic V-ATPase inhibitors is often not known. Novel small molecules with defined mechanism of inhibition against V-ATPase are needed to evaluate the therapeutic potential of V-ATPase inhibitors in human diseases.